Alternative splicing is tightly regulated so that each cell can express an appropriate protein repertoire in the appropriate developmental and physiological contexts. In fact, there are numerous examples of human disease gene mutations in which the mutations cause aberrant splicing of the affected transcripts. The long term objectives of this application include elucidation of the biochemical bases that underlie different alternative splicing events, and evaluation of impact of individual alternative splicing regulators at the cellular and organismal level. Specific aims are to: 1. Purify and characterize single pre-mRNP complexes assembled in vivo. The protein components of the pre-mRNP complexes undergoing different alternative splicing events in the cells will be directly and comprehensively analyzed. The model alternative splicing substrates, such as Drosophila P element pre mRNA containing the third intron (IVS3), will be expressed in vivo as a fusion with the recently developed tandem RNA tags. The in vivo assembled pre-mRNP complexes will then be affinity-purified and the associated proteins will be identified by mass spectrometry. Comparison between two alternatively spliced forms will be achieved by (a) using wild-type (IVS3 splicing silenced) and mutant (IVS3 splicing activated) substrates, and (b) using normal (IVS3 splicing silenced) and modified (IVS3 splicing activated) cells in which the known cell-type specific splicing regulators such as PSI are removed by RNAi. 2. Purify and characterize the splicing silencer complex assembled in vitro. This Aim complements the Aim 1, and is conducted with more conventional in vitro system. The wild-type or mutant P element pre mRNA substrate will be synthesized with an RNA-tag, and pre-mRNP complexes will be assembled in vitro and affinity-purified. The protein components will be determined by mass spectrometry. The in vivo functions of the newly identified splicing regulators will be investigated by removing them (RNAi) and monitoring alteration of splicing patterns of the transcripts (RT-PCR or splice-junction microarrays) in cell culture. 3. Investigate the physiological functions of alternative splicing regulator PSI. In the mutant expressing truncated splicing regulator PSI, females are normal but males exhibit spermatogenesis and behavioral defects. Using the splice-junction microarrays, genome-wide alternative splicing patterns will be compared between the wild-type and mutant testes and brains to understand the biological significance of this splicing regulator. These studies will provide general understanding of how aberrant splicing could cause disease phenotypes. The biochemical insights obtained here may also allow novel therapeutic interventions.